Prolongation of clotting time of blood



CLOTTING TIME IN SECONDS y 0, 1968 R. L. COLESCOTT ETAL 3,395,222

PROLONGATION OF CLOTTING TIME OF BLOOD Filed March .1, 1966 5sheetsrsheet l 30o KO 29|OO I 1 QS I N wig ANTICOAGULANT EFFECTS OFHOEPARIN HEPARIN AND PHOSPHOLIPID SINGLY AND m COMBINATION KO29|OO $252.04 UNITS fHEPARIN HEPARTJ USP UNITS I00 .02 .04 .06 .08 .l v.l2

M68 OF PHOSPHOLIPID lNI/ENTORS ROBERT L. COLESCOTT ARCH/E L. CALDWELLJR.

CLOTTING TIME IN SECONDS July 30, 1968 R. COLESCOTT ETAL 3,395,222

PROLONGATION OF CLOTTING TIME OF BLOOD Filed March 11, 1966 5 Shets-Sheet 2 ACTUAL EFFECT ADDlTIVE SEASIKWUS QI I P'P s QF gFfifiTTglgFPHOSPHOLIPID D8 UMT 0 EPARlN PLUS IOO PHOSPHOLIPI l ACTUAL EFFECT OF .04UNIT OF HEPARlN PLUS PHOSPHOLIPID IN VITRO ANTICOAGULANT SYNERGISMBETWEEN HEPARIN AND PHOSPHOLlPlD .I .2 .3 .4 .5 M68 OF PHOSPHOLIPIDINVENTORS ROBERT L. COLESCOTT ARCH/E L. CALDWELL, JR.

y 1968 R, v COLESCOTT ETAL 3,395,222

PROLONGATION OF CLOTTING TIME OF BLOOD Filed March .1, 1966 3Sheets-Sheet 3 NTICOAIGULANT EFFECTS OF H EPARIN AND bLlPlDS SINGLY ANDIN COMBINATION.

350 HEPARlN-PHOSPHOLIPID COMBINATIONS F TABLE FIVE 0 z I: 200 O J U EADDITIVE EFFECT I 3 USP UNITS: MG PHOSPHOLIPID/2Z 5 I2 I Z I0 20 3O 4O5O 6O 7O I00 HEPARIN IN USP UNITS/KG OF ANIMALS BODY WEIGHTPHOSPHOLIPIDS IN MG/KG ANIMAL BODY WEIGHT INVENTORS. ROBERTL. COL ESCOTTARCH/E L. CALDWELL, JR.

United States Patent ABSTRACT OF THE DISCLOSURE Preparations forprolonging the clotting time of blood comprising a synergisticcombination of heparin and an anti-thromboplastic phospholipid; and amethod of ad ministering such preparations to human beings and otherwarm blooded animals.

This application is in part a continuation of our copending applicationSer. No. 168,016, filed J an. 19, 1962, now abandoned and describes aninvention which relates to the prolongation of clotting time in mammals,and more particularly to the use of heparin and a phospholipid incombination for prolonging the blood clotting time in human beings andother warm blooded animals.

An object of the invention is to provide a preparation and method forprolonging the clotting time of blood. A further object is to providefor the use in combination of heparin and an anti-thromboplasticphospholipid which when administered to mammals, act synergisticallywith each other to increase the clotting time of the mammals blood.These and still further objects as shall hereinafter appear arefulfilled by the present invention as will be readily discerned from aconsideration of the following detailed description of embodimentsexemplifying the salient features hereof, particularly in view of theaccompanying drawings in which:

FIG. 1 is a graph showing in vitro anticoagulant effects of heparin(soluble, 1000 U.S.P. Units/cc.) and liver phospholipid K 029100(prepared according to Dailey, U.S. Patent No. 3,089,820), singly and incombination, wherein the ordinant represents clotting time in secondsand the abscissa represents units of heparin (upper scale) andmilligrams of phospholipid (lower scale);

FIG. 2 is a graph showing an in vitro comparison between additive effectof heparin-phospholipid (calculated from the values of FIG. 1) when .04and .08 units of heparin are added to the amount of phospolipidindicated by the abscissa of the graph, and the actual effect of heparinplus phospholipid (observed) when .04 and .08 U.S.P. units of heparinare admixed with the amount of phospholipid indicated by the abscissa ofthe graph; the ordinant of the graph representing clotting time inseconds; and

FIG. '3 is a graph showing the in vivo effect, measured as increase inthe clotting time in minutes (ordinant), of admixed heparin andphospholipid, heparin being reported in U.S.P. units per kilogram ofanimal body weight and phospholipid being reported in milligrams perkilogram of animal body weight (abscissa), both singly and incombination (the Weight ratio of heparin to phospholipid in theadmixture being 1 to 100) as well as providing a comparison between themeasured effect of the combination (top curve) and the calculatedadditive effect of heparin plus phospholipid (dotted line).

ice

Phospholipids and their effect on blood coagulation are well known andmay be classified as follows: (1) procoagulant (thromboplastic)phospholipidsphospholipids which accelerate the clotting rate of blood,i.e., shorten the clotting time; (2) neutral phospholipidsphospholipidswhich neither accelerate nor delay the clotting rate, i.e., have noeffect on clotting time; and (3) anticoagulant (anti thromboplastic)phospholipids phospholipids which delay the clotting rate, i.e., prolongthe clotting time.

For the practice of our invention, we prefer to employ anticoagulant(anti-thromboplastic) phospholipids for use in combination with heparinwhich is an anticoagulant substance. The combination of heparin and suchphospholipids produces an effect upon the coagulation of blood both invivo and in vitro greater than the sum of effects of the separateagents. The heparin and phospholipids may be premixed and the mixtureadministered parenterally or the heparin may be administeredparenterally and the phospholipids administered orally. In addition, themix ture may be added to an in vitro system. The synergistic effect canalso be produced by treating the patient with phospholipid and heparinsimultaneously or successively and the agents may be introduced by thesame or different routes provided both substances are present in thecirculating blood simultaneously. It is found desirable to havesufficient heparin present to provide a heparin .to phospholipid weightratio of at least about 1:750 up to about 1:50.

As indicated, the synergistic effect produced by the simultaneous orsuccessive administration of heparin and the anti-coagulant phospholipidis illustrated by the drawings. The in vitro demonstration of thesynergistic effect is shown by FIGS. 1 and 2. It can be seen from FIG. 1that the slope of the curve indicating the prolongation of coagulationtime by the mixture is different from the slopes of the curves foreither component alone.

In FIG. 2, it is also apparent the slope of the curve describing theeffect of the synergistic mixture is different from the slope of thecurve that would have been obtained if the effect of the two materialshad been only additive.

To further aid in the understanding by the in vitro aspects of thisinvention, and not by way of limitation, reference is made to thefollowing examples:

Example I In vitro determinations were performed by conducting clottingtime tests in 12 x mm. culture tubes maintained at 37 C. in a waterbath. Into each tube, we placed 0.2 ml. of Diagnostic Plasma(Warner-Chilcott), 0.1 ml. of Platelet Factor (Warner-Chilcott),prepared by diluting one vial to 5 ml. with 0.05 M imidazole buffer atpH 7.4; 0.1 ml. of imidazole buffer containing the material to betested; and 0.1 ml. of 0.05 M calcium chloride solution. The calciumchloride was added last and a stop was started at the moment of calciumchloride addition. Clotting was detected by gently tilting the tubeevery ten seconds. The time elapsing from the addition of the calciumchloride until clotting occurred was recorded.

The results obtained conducting the foregoing on a control, i.e., bloodwith no added reparin or phospholipid, and on blood with additions ofvaried amounts of heparin and/ or liver phospholipid (coded K 029100 andprepared according to the teaching of Dailey et al., U.S. 3,089,820, May14, 1963) are reported in Table 1 below.

TABLE 1 Amount of Clotting Increase in Amount of Heparin Phospholipid*Time Clotting (U.S.P. units) (milligrams) (seconds) Time (seconds) Liverphospholipid K 029100.

(Control).

It may be observed from the data of Table 1 that the clotting timeincreases with heparin and phospholipid in combination are greater thanwould be predicted by simple addition of the results when each substancewas tested alone.

Example II Further in vitro determinations were conducted using the samegeneral protocol described in Example I except that the culture tubeswere kept in an air bath at 37 C. and that clotting time was detected bygently dipping a wire loop through the solution in the tube atapproximate one second intervals. The results of these in vitrodeterminations are reported in Table 2, the phospholipid here used beinga liver phospholipid code K 136037D which was prepared according to theprocedure described in Adams and Colescott co-pending application Ser.No. 167,437, filed Jan. 19, 1963, now abandoned (Canadian Patent742,715, issued Sept. 13, 1966).

thetic with pentobarbital sodium throughout the experiment. Theanesthetic was administered intravenously by way of the marginal earvein in a dosage of 25 mg. per kilogram of body weight. Blood sampleswere obtained by means of a polyethylene cannula surgically placed inthe left cartoid artery. The blood was allowed to flow directly from thecannula into the sample cup of the thrombelastograph. Two blood sampleswere obtained from each rabbit, the first being taken prior to treatmentand the second being taken fifteen (15) minutes after treatment with thematerial being tested.

The material to be tested (phospholipid, heparin, or combinations ofheparin and phospholipid) were dissolved in isotonic solution and theconcentration of the test material was varied in such manner that thedosage was always given in a total volume of one milliliter per kilogrambody weight.

All test solutions were administered by way of the marginal ear vein.

The control clotting time determined by calculating the arithmetic meansof the pretreatment clotting times was found to be 23.4 minutes (mean ofeighty-four rabbits).

As would be expected, clotting time increased progressively withincreasingly larger doses of heparin as shown in Table 3.

TABLE 3 Pretreat- Posttreat- Increase in Dose of Heparin Number meritmerit Clotting (U.S.P. units lotting Clotting Time per kg.) Rabbits TimeTime (min.) 30 (min.) (min.)

Similarly, the clotting time increased with increasingly large doses ofphospholipid as is shown by the data shown in Table 4.

40 TABLE 4 TABLE 2 Amount of Clotting Increase in I Pretrcat- Posttreat-Increase in A t of Heparin Phospholipid Ti Cl tt Dose of PliospholipidNumber mer t ment Clotting (U.S.P. units) (milligrams) (seconds) Time g/it) CIQttmg Clotting Time (seconds) Rabbits Time Time (min.)

(min.) (min.)

0 115 O 0 110 5 3 23. 9 25. 4 1. 5 0 15 ('2 22. 4 25. 4 3. 1 0 30 G 24.4 38. 8 14. 4 0 189 74 6 23. 8 41. 5 17. 7 0. 05 129 14 6 25. 4 87. 261. 8 0. l 147 32 [i 23. 4 49. B 26. 4 0. 2 160 45 6 24. 2 57. 6 33. 40. 3 191 76 6 24. l 79. 6 55. 5 O. 05 40 O. 1 192 77 8' 3 g? It would beordinarily expected that administration of 0. 1 240 235 a mixture ofheparin and phospholipid would produce an Liver phospholipid K 136037D.1 Control.

It may again be noted that the clotting time increases obtained withheparin and phospholipid in combination are greater than would bepredicted by simple addition of the results obtained when each substancewas tested alone.

The data reported above have been in part plotted and are shown in FIGS.1 and 2 of the drawings.

To aid in a further understanding of the in vivo aspects of thisinvention, and not by way of limitation, reference shall now be made tothe following examples.

Example III In vivo experiments with rabbits, a laboratory animal havingaccepted correlation to human blood coagulation properties, wereconducted as follows: Before treatment of the animals, clotting timedeterminations were carried out on arterial blood (whole blood) by meansof the thrombelastographic technique (P. De Nicola, Thrombelastography,Chas. C. Thomas, Publisher, Springfield, Ill., 1967). All of the rabbitswere maintained under anesincrease in clotting time which could bepredicted by simple addition of the clotting time increases produced byeach substance alone. As an example, from the above results it would bepredicted that administration of a mixture of heparin and phospholipidsuch, that each animal received 20 U.S.P. units/kg. of heparin and 20mg./kg. of phospholipid would result in an increase in clotting time of27.9 minutes (14.4 minutes+13.5 minutes). In actual practice it wasfound that the clotting time increases resulting from the administrationof mixtures of heparin and phospholipid were considerably greater thanwould be predicted. The actual results obtained are reported in Table 5below.

TABLE 5 Pretreat- Posttreat- Dose 0! Dose of The results of Table 5clearly show that heparin and phospholipid act together synergistically.

The effects of the tested materials were determined by calculating theincrease in clotting time of the posttre'atment samples over thepretreatment samples, as indicated. These increases are plotted in FIG.3. Examination of the drawing reveals graphically that the increases inclotting time caused by our combination of heparin and phospholipid isgreater than could be expected on the basis of arithmetically additiveelfects and clearly demonstrate synergism between heparin andphospholipids in vivo. The phospholipid used in this determination wasliver phospholipid K 029100, previously identified.

Still further understanding of the present invention can be garneredfrom a consideration of the following examples.

Example IV A typical method of preparing a soybean phosphatide may bedescribed as follows:

500 grams of soybean phosphatide concentrate (A. E. Staley & Co. STA-SOLUR) was extracted three times with 3,000 ml. portions of acetone. Theresidue was vacuum dried and then extracted twice with 3,000 ml.portions of anhydrous denatured ethyl alcohol. The residue was thendissolved in about 400 ml. of chloroform and precipitated by pouringinto 2,000 ml. of acetone. The precipitate was collected on a Biichnerfunnel and dried under vacuum. The yield of this alcohol-insoluble soyaphosphatide was 114 grams. 100 grams of this material were then placedin an open pan in an oven at 70 C. for two weeks to render the soybeanphosphatide highly anti-thromboplastic.

The heparin was then pre-mixed with the anti-thromboplastic phosphatideand the preparation administered parenteral to rabbits as describedabove in connection with FIG. 3.

Example V The soybean phosphatide, prepared according to Example IV, wasused in performing the in vivo determination reported in Example III.

The data reflecting the use of heparin alone is shown in The results areconsistent with our expectations and previous results. I

The data reflecting the use of the soybean phosphatide of Example IValone are reported in Table 7.

TABLE 7 Pretreat- Posttreat- Number ment Olotment Clot Dose of Dose ofHeparin Phpspho- Increase in (U.S.P. lipid" of ting Time ting TimeClotting (mg. /kg.) Rabbits (min.) (min.) Time (min.)

*Soybean phospholipid K 075299.

These results are also consistent with our expectations and our previousresults.

Finally the data showing the in vivo synergism of combinations ofheparin and phospholipid are reported in Table 8.

TABLE 8 Dose of Dose of Pretreat- Posttreat- Heparin Phospho- Numberment Olotment Clot- Increase in (U.S.P lipid of ting Time ting TimeClotting (mg/kg.) Rabbits (min.) (min.) Time (min.)

Soybean phospholipid K 075299.

It should be understood from the foregoing that heparin and phospholipidmay be employed in any desired proportions, since the proportionsthereof are not critical. The addition to heparin of substantially anyamount of the anti-thromboplastic phospholipid is effective in givingthe results which have been described above.

While the heparin and phospholipid are in usual practice bothadministered parenterally, the synergistic effect will still be obtainedregardless of route of administration provided both substances arepresent in the circulating blood simultaneously. Thus, phospholipid canbe administered orally as well. Of course, heparin administration wouldusually be by parenteral administration.

It should be understood that in correlating the weight of phospholipidto the weight of heparin, when heparin is presented as U.S.P. units,notice should be made of the relationship that heparin containsapproximately U.S.P. units per milligram. Thus, weight ratios of, forinstance, 0.04 U.S.P. units of heparin to 0.3 milligram of phospholipidmay be readily calculated using but ordinary mathematical skills.

The term anti-coagulant phospholipid as herein used is known to the art.The two principal sources of anticoagulant phospholipid are animals andplants which may be characteristically illustrated by mammalian tissue,e.g., liver and soyabean. See: Activation of Purified Prothrombin toAutoprothrombin I or Autoprothrombin II (Platelet Cofactor II) orAutoprothrombin II-A, Mammen, E.F., Thomas, W. R., and Seegers, W. H.,Thromb. Diath. haem. 'vol. 5, p. 280 (1960) and particularly Fig. 1 andp. 218.

It should also be understood that we have herein defined our synergisticcombinations in terms of minimal proportions which are capable ofextending clotting time well beyond normal desires. To further extendthese proportions would render the blood virtually uncoagulable andwould serve no useful purpose.

From the foregoing description and examples it can be seen that new andunexpected benefits have been obtained by our discovery of synergisticcombinations of heparin and phospholipids for prolonging the clottingtime of blood which, in addition, to fulfilling all of the aforestatedobjectives in a remarkably unexpected manner, is intended to includesuch modifications and applications and alterations as may readily occurto the artisan confronted with this disclosure as within the spirithereof, especially as it is defined by the scope of the claims appendedhereto.

What is claimed is:

1. A method for prolonging the clotting time of blood comprisingadministering systemically to human beings and other warm bloodedanimals heparin and anti-coagulant phospholipid in a weight ratio of atleast about 1 part heparin to 750 parts phospholipid up to about onepart heparin to about 50 parts phospholipid for concurrent engagementwith said blood, said heparin being administered parenterally.

2. The method according to claim 1 in which said heparin and saidanti-coagulant phospholipid are administered simultaneously.

3. The method according to claim 2 in which said heparin and saidphospholipid are administered parenterally.

4. The method according to claim 1 in which said heparin and saidanti-coagulant are admixed prior to said administration to form anadmixture having a heparin to phospholipid weight ratio of about 1:100.

5. The method according to claim 3 in which said parenteraladministration is intravenously.

6. A preparation for the .in vivo prolongation of blood clotting timecomprising in synergistic combination heparin and an anti-coagulantphospholipid, said heparin being present in sufficient quantity toprovide a heparin to phospholipid weight ratio of at least about 1:750up to about 1:50.

7. A preparation according to claim 6 in which said weight ratio isabout 1:100.

8. A preparation according to claim 6 containing at References CitedUNITED STATES PATENTS 6/1963 Dailey 167-74.6

OTHER REFERENCES ALBERT T. MEYERS, Primary Examiner.

least about 0.01 U.S.P. units of heparin per milligram of 15 A, FAGELSONAssistant E i phospholipid.

